Nonalcoholic steatohepatitis (NASH) is prevalent in over 70% of the obese and type II diabetes mellitus (T2DM) patients and is a leading cause of liver transplantation. Hepatic insulin resistance and inflammation mirror alterations in mitochondrial oxidative flux (beta-oxidation, tri-carboxylic acid [TCA] cycle and mitochondrial respiration), in rodent models and humans with simple steatosis. This proposal will identify new mechanisms leading to dysfunctional mitochondrial oxidative flux and development of NASH. The proposal will test the hypothesis that the severity of hepatocyte inflammation and reactive oxygen species (ROS) generation will be proportional to the rates of mitochondrial oxidative flux. Thus, attenuation of oxidative flux will provide a promising strategy to alleviate inflammation and oxidative stress in NASH and T2DM. Aim 1 will test whether altered mitochondrial oxidative flux during the transition from simple steatosis to NASH parallel an increase in ROS production, inflammation and oxidative stress. This will be tested in a mouse model fed high fructose, high trans-fat diet which gradually transitions from simple steatosis to NASH, closely resembling human disease. Aim 2 will investigate a novel mechanism by which chronic elevation of branched chain amino acids (BCAAs) during hepatic insulin resistance will disrupt mitochondrial oxidative flux and sustain lipogenesis. Aim 3 will utilize a novel mouse model with a clinically relevant polymorphism in the NADH dehydrogenase subunit 2 (mt-Nd2A) allele. This unique animal model will help identify whether the upregulated antioxidant defense resulting from the beneficial effects of the mt-Nd2A allele will attenuate mitochondrial oxidative dysfunction in NASH. State-of-the-art metabolic profiling techniques in nuclear magnetic resonance and mass spectrometry will be combined with measures of ROS production and oxidative stress in liver. Direct measurements of oxidative flux through the TCA cycle relative to ATP turnover in the liver will result in a novel index of mitochondrial coupling efficiency, which will have high translational value for human studies. In conclusion, identifying key mechanisms to attenuate mitochondrial oxidative flux will provide a better paradigm to treat NASH and decrease unregulated gluconeogenesis in T2DM.